Silencing of tgf-beta receptor type II expression by sirna

ABSTRACT

The present application is directed to siRNA-based silencing of the type II receptor of TGFβ. siRNAs that target this receptor abrogate the receptor protein and transcript, TGFβ-mediated processes such as fibronectin assembly and cell migration also are inhibited and the molecules of the invention are efficacious in reducing the inflammatory response and matrix deposition. These findings show that siRNAs can be successfully delivered both in vitro and in vivo to regulate the TGFβ type II receptor level and modulate wound response. Methods and compositions exploiting the findings of the present invention have a wide-ranging application, extending from treatment of disorders of the eye to other organs and tissues throughout the body.

FIELD OF THE INVENTION

The present invention is directed to methods and compositions forsilencing transforming growth factor beta type II receptor (TGFβRII)expression. More particularly the present invention describes methodsand compositions for reducing such expression using small interferingRNA (siRNA) molecules.

BACKGROUND OF THE INVENTION

Transforming growth factor-β (TGFβ) comprises a family of structurallyrelated multifunctional cytokines. They have a wide variety ofbiological actions, including cell growth, differentiation, apoptosis,fibrogenesis and angiogenesis. (Massague et al., Cancer Surv. 12,81-103, (1992), Piek et al., FASEB J. 13, 2105-2124, (1999), Border &Noble N. Engl. J. Med. 331, 1286-1292 (1994); Govinda and Bhoola,Pharmacol. Ther. 98:257-265 (2003); Cusiefen et al., Cornea 19:526-533;Sakimoto et al., Gene Therapy 7:1915-1924 (2000)) TGFβ is typicallysecreted in a biologically latent form. It is activated through acomplex process of proteolytic activation and dissociation of latencyprotein subunits. (Massague, Annu. Rev. Biochem. 67, 753-791 (1998)).

The mechanism of action of TGFβ is mediated by its binding to receptorsknown as TGFβ receptors, types I, II and III. Receptors I and II aretransmembrane glycoproteins of 55 and 70 kDa shown to be important insignal transduction. The TGFβ ligand binding site for these receptors isextracellular. The mechanism by which the signaling is thought to beachieved is via activation of phosphorylation of transcription factorsknown as Smads. (Massague & Wotton, EMBO J. 19, 1745-1754 (1999)).

TGFβ has emerged as a key component of the fibrogenic response towounding and is upregulated during many different types of wound healingin tissues such as the eye, liver, and skin. (Border & Noble, N. Engl.J. Med. 331, 1286-1292 (1994), Connor et al., J. Clin. Invest. 83,1661-1666 (1989), McCormick et al., J Immunol. 163, 5693-5699 (1999),Shah et al., J. Cell Sci. 108, 985-1002 (1995)). In the eye, of thethree human isoforms (TGFβ1, TGFβ2, and TGFβ3), TGFβ2 is the predominantone. (Lutty et al., Invest. Ophthalmol. Vis. Sci. 34, 477-487 (1993),Pasquale et al., Invest. Ophthalmol. Vis. Sci. 34, 23-30 (1993)). TGFβshave been implicated in several scarring processes includingproliferative vitreoretinopathy, (Kon et al., Invest. Ophthalmol. Vis.Sci. 40, 705-712 (1999)), cataract formation, (Hales et al., Invest.Ophthalmol. Vis. Sci. 36, 1709-1713 (1989)), corneal opacities, (Chen etal., Invest. Ophthalmol. Vis. Sci. 41, 4108-4116 (2000)), andconjunctival wound healing, (Cordeiro, Clin. Sci. 104, 181-187 (2003))especially that occurring after filtration surgery for a major blindingdisease, glaucoma. In addition, TGFβ in conjunction with connectivetissue growth factor (CTGF) has an important role in angiogenesis (Abreuet al., Nature Cell Biol. 4:599-604 (2002)). Furthermore, recent studieshave shown that TGF may actually be involved in the pathogenesis ofprimary open angle glaucoma (Inatani et al., Graefes Arch. Clin. Exp.Ophthalmol. 239(2):109-13, 2001; Ochiai et al., Jap. J. Ophthalmol.46(3):249-53, 2002; Gattanka et al., Invest. Ophthalmol. Vis. Sci.45(1):153-8, 2004).

In glaucoma filtration surgery, excessive postoperative scarring at thewound site significantly reduces surgical success. (Migdal et al,Ophthalmology 101, 1651-1656 (1994), Addicks et al., Ophthalmol. 101,795-798 (1983)). Although anti-scarring agents such as mitomycin-C and5-fluorouracil could help prevent postsurgical scarring and improveglaucoma surgical outcome, (Khaw et al., Arch. Ophthalmol. 111, 263-267(1993), Cordeiro et al., Invest. Ophthalmol. Vis. Sci. 40, 1975-1982(1999)) they do so by causing widespread fibroblast cell death and areassociated with severe and potentially blinding complications. (Crowstonet al. 449-454 (1998), Stamper et al., Am. J. Ophthalmol. 114, 544-553(1992)). In light of the role of TGFβ in the wound repair process,alternative strategies (Codeiro, Prog. Retin. Eye Res. 21, 75-89 (2002))such as antibodies (Cordeior et al., Invest. Ophthalmol. Vis. Sci. 40,2225-2234 (1999), Mead et al., Invest. Ophthalmol. Vis. Sci. 44,3394-3401 (2003)) to TGFβ and antisense oligonucleotides (Cordeior, etal., Gene Therapy 10, 59-70 (2003)) have been used to block TGFβ action.However these techniques remain inadequate for the treatment of thedebilitating scarification that occurs in many glaucoma. For example,use of antisense therapy is poorly effective in treating variousdisorders because antisense molecules are known to induce an interferonresponse in the patient. Use of antibody-based therapies are marred bythe need to generate specific antibodies against particular epitopes ofa given antigen. Thus, there remains a need to identify new methods ofintervening in disorders that result from an over-expression or evenmere presence of TGFβ type II receptor.

SUMMARY OF THE INVENTION

The present invention is directed to the use of siRNA both in vitro andin vivo to regulate the TGFβ type II receptor (TGFβ RII) level andmodulate wound responses and angiogenesis in a mammal. The RNAinterference-based methods of the present invention have a wide-rangingapplication, extending from the eye to other organs and tissuesthroughout the body.

In certain embodiments, the invention is directed to methods andcompositions for promoting wound healing, reducing fibrosis and/orreducing angiogenesis in a mammal by administering to the mammal acomposition comprising siRNA molecules that target the type II receptorof TGFβ.

The siRNA molecules of the present invention may be delivered, in atherapeutically effective amount, locally at the site of the wound oralternatively may be administered systemically. In certain embodiments,therapeutically effective siRNA compositions may be administered aloneor alternatively, the siRNA molecule-based therapeutic compositions maybe administered as part of a therapeutic regimen that comprises otherwound-healing compositions.

In particularly preferred embodiments, the disorder to be treated by thesiRNA based therapeutic compositions of the present invention isglaucoma. However, it should be understood that the siRNA compositionsof the present invention may be used in the treatment of any disorder inwhich signaling through the TGFβ type II receptor is implicated. Inaddition to glaucoma filtration surgery, the compositions of the presentinvention may be used to promote healing, with a reduction in scarring,of any other ophthalmic surgery, which may include but is not limitedto, cataract extraction, with or without lens replacement; cornealtransplants, to treat viral infection or penetrating keratoplasty (PKP);and radial keratotomy and other types of surgery to correct refraction.The compositions and methods of the invention also may be used to treatocular disorders such as, e.g., retinal wounds such as retinaldetachments and tears, retinal vacuolar disorder, retinalneovascularization, diabetic retinopathy, corneal wounds such as cornealepithelial wounds, corneal neovascularization, corneal ulcers, macularholes, macular degeneration, secondary cataracts, corneal disease, dryeye/Sjogren's syndrome and uveitis. These disorders include woundhealing disorders, proliferative disorders, anti-degenerative disordersand anti-angiogenesis disorders that effect the eye.

In each of the above methods, the method involves administering to themammal an amount of the siRNA composition in an amount effective tostabilize or improve vision. Retinal disorders, which are characterizedby increased connective or fibrous tissue, also may be treated usingmethods which comprise the steps of removing the vitreous humor from theeye; removing the epiretinal membrane, if present, from the eye; andadministering a composition comprising the siRNA compositions of theinvention by cannula to place the therapeutic composition immediatelyover the portion of the retina requiring treatment.

In certain other embodiments, the siRNA composition may be administeredby intraocular injection or by application to the cornea. Such cornealapplication may be achieved using eye drops or a timed release capsuleplaced in the cul de sac.

In another embodiment, there is provided a method for treating a mammalfor ocular neovascularization, said method comprising administering to amammal an effective amount of the siRNA compositions of the presentinvention.

Other non-ocular disorders that may be treated using the siRNA-basedmethods of the present invention include but are not limited tofibroproliferative disorders such as those selected from the groupconsisting of diabetic nephropathy, glomerulonephritis, proliferativevitreoretinopathy, liver cirrhosis, biliary fibrosis, and myelofibrosis,post-radiation fibrosis. Connective tissue disorders such as rheumatoidarthritis, scleroderma, myelofibrosis, and hepatic, and pulmonaryfibrosis also may be treated. Disorders involving defective T-cellresponse, such as trypanosomal infection or viral infections such ashuman immunosuppression virus, human T cell lymphotropic virus,lymphocytic choroiomeningitis virus and hepatitis may be treated. siRNAmethods may be used to treat patients with cancer, including patientswith prostate cancer, ovarian cancer, plasmacytoma and glioblastoma.siRNA may be used to treat patient with collagen vascular diseases suchas progressive systemic sclerosis (PSS), polymyositis, dermatomyosistisand systemic lupus erythamaosus.

In addition, siRNA-based methods may be used to treat wounds other thanthose induce by ocular trauma, disorders or surgery. Surgical incisionsin general, trauma-induced lacerations, fibrosis due to radiationtherapy and wounds involving the peritoneum may be treated. Scarringresulting from restenosis of blood vessels, hypertrophic scars andkeloids may also be treated with siRNA methods.

Particularly preferred siRNA molecules include 21-23 bases. Fourspecific sequences for the TGFβRII siRNA were derived from the humanTGFβRII sequence (Genbank Accession Number: M85079) and were designatedas NK1, NK2, SS1 and SS2. The target sequences (5′ to 3′) are set out asbelow, with the position of the first nucleotide in the human TGFβIIreceptor sequence (from M85079) shown in parenthesis. The correspondingcommercially synthesized siRNA duplexes are also set out below: TargetSequence 5′ to 3′ Nucleotide number in parenthesis siRNA duplex NK1(529) UCCUGCAUGAGCAACUGCAdTdT AATCCTGCATGAGCAACTGCAdTdTAGGACGUACUCGUUGACGU (SEQ ID NO: 1) (SEQ ID NOS: 5-6) NK2 (1113)GGCCAAGCUGAAGCAGAACdTdT AAGGCCAAGCTGAAGCAGAAC dTdTCCGGUUCGACUUCGUCUUG(SEQ ID NO: 2) (SEQ ID NOS: 7-8) SS1 (1253) GCAUGAGAACAUACUCCAGdTdTAGCATGAGAACATACTCCAG dTdTCGUACUCUUGUAUGAGGUC (SEQ ID NO: 3) (SEQ ID NO:9-10) SS2 (948) GACGCGGAAGCUCAUGGAGdTdT AAGACGCGGAAGCTCATGGAGdTdTCUGCGCCUUCGAGUACCUC (SEQ ID NO: 4) (SEQ ID NO: 11-12)

It should be understood that those of skill in the art will be able toproduce additional siRNA molecules surrounding positions 529, 1113, 1253and 948 of the human TGFβRII gene sequence at Genbank Accession Number:M85079. It should be understood that the siRNA molecules of theinvention may be conveniently formulated into pharmaceuticalformulations using methods known to those of skill in the art. Suchpharmaceutical compositions also may comprise other non-siRNA basedtherapeutic agents for the therapeutic intervention of the particulardisorder being treated. Other wound healing compositions includeanti-cancer drugs Mitomycin and 5-fluorouracil, agaricus bisporuslectin, metallocomplexes such as zinc-desferrioxaminde orgallium-desferrioxamine, methyl xanthine derivatives such aspentoxifylline, collagen-based sealants such as GE Amidon Oxyde, agentsthat inhibit fibroblast growth factors and connective tissue growthfactor, and matrix metalloproteinase inhibitors such as ilomastat. Otheranti-angiogenic agents include inhibitors of vascular endothelial growthfactor (VEGF) and antiangiogenic steroids. Inhibitors of VEGF includesiRNA molecules targeting VEGF or its receptor.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, because various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further illustrate aspects of the present invention. Theinvention may be better understood by reference to the drawings incombination with the detailed description of the specific embodimentspresented herein.

FIG. 1. Inhibition of TGFβ type II receptor expression by siRNA.Immunofluorescence analysis of human corneal fibroblasts untreated(1^(st) row), or treated with scrambled siRNA (2^(nd) row) or 100 nM NK1(3^(rd) and 4^(th) rows) was performed to visualize TGFβRII receptorexpression (left column). Staining of nuclei using DAPI stain is shownin the right column. Note the large reduction in staining of cellstreated with NK1 siRNA at 48 (3^(rd) row, left column) and 72 h (4^(th)row, left column) compared to control cells (1^(st) and 2^(nd) rows,left column).

FIG. 2. Suppression of TGFβI type II receptor protein expression bysiRNA in corneal fibroblasts. Lysates from human corneal fibroblaststreated with different concentrations of TGFβRII receptor siRNA orcontrol, scrambled siRNA for 16 (top panel) or 48 hours (bottom panel)were separated on 10% SDS-polyacrylamide gels and immunoblotted with aTGFβRII receptor antibody. Lane 1 contains lysate from cells incubatedonly with TransIT-TKO reagent (no siRNA). Lanes 2 and 8 contain lysatesfrom cells treated with 100 nM scrambled siRNA. Lanes 3, 4, 9 and 10contain lysates of cells treated with NK1 siRNA at a final concentrationof 50 (lanes 3 and 9) or 100 nM (lanes 4 and 10). Lanes 5, 6, 11 and 12contain lysates of cells treated with SS1 siRNA at 50 (lanes 5 and 11)or 100 nM (lanes 6 and 12). In lane 7, the TGFβRII receptor antibody waspreincubated with antigenic peptide before probing the normal celllysate. Similar amounts of total protein were loaded in each lane.

FIG. 3A-3G provides target sequences in the TGFβ type II receptorsequence and the corresponding siRNA molecule sequences. The nucleotidenumbers refer to the location in the type II TGF-β receptor sequence(Genbank Accession Number: M85079). The GC content refers to the contentof guanine and cytosines (GC) within the target sequence.

FIG. 4. Inhibition of TGFβRII using siRNA on HUVEC cells. Human umbicalvein endothelial cells (HUVEC) were plated at 3×10⁻⁵ and allowed to growinto confluent monolayers. Following day the cells were treated with (a)control (TKO reagent only), (b) scrambled (c) NK1 siRNAoligonucleotides, (d) SS1 siRNA oligonucleotides, all in the TKOreagent. Images were taken 48 hours post RNAi treatment. e, f, g, and hare corresponding DAPI nuclear staining of the cells in panels a, b. c,and d respectively. Scale bar is 10 microns.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

There is a need to develop new therapies for reducing scarring thatresult during wound healing. TGFβ is known to be involved in thefibrogenic response in wound healing, and inhibition of TGFβ-inducedactivities may be therapeutically effective for reducing fibrosis andscarring. The present invention provides specific siRNA compositions foruse in methods of promoting wound healing and for reducing scarring as aresult of wound healing. In addition, the present invention providesspecific siRNA compounds for use in methods of inhibiting angiogenesis.These compositions are described in further detail herein.

Definitions

The term RNA interference (RNAi) refers to post-transcriptional genesilencing induced by the introduction of double stranded RNA.

The term small interfering RNAs (siRNAs) refers to nucleotides of 19-23bases in length which incorporate into an RNA-induced silencing complexin order to guide the complex to homologous endogenous mRNA for cleavageand degradation of TGFβRII and that mRNA.

The term transforming growth factor (TGFβ) refers to a family of peptidegrowth factors including five member, numbered 1 through 5.

The term TGFβ receptors refers to cell surface proteins, of which three(Type I, Type II and Type III) are known in mammals. The TGFβ type IIreceptor (TGFβRII) is a membrane bound protein with an intracellulardomain, a transmembrane domain and extracellular domain that binds toTGFβ. As reviewed in Massague et al., Annu. Rev. Biochem. 67: 753-791,(1998) incorporated herein by reference.

The term therapeutically effective amount refers the amount of a siRNAmolecule which effectively suppresses expression of the TGFβRII proteinin a mammal in need.

Role of TGFβ Family in Wound Healing

Transforming growth factor-β (TGFβ) family of cytokines is an importantmediator in the wound healing process in various tissues. In the eye,TGFβ has been implicated in the corneal haze and scarring at the woundsite following glaucoma surgery. TGFβ has also been associated withdiabetic retinopathy, proliferative vitreoretinopathy and maculardegeneration. The inventors designed small interfering RNAs (siRNAs)targeting the type II receptor of TGFβ and found that these RNAfragments were effective in abrogating the receptor protein andtranscript in cultured human corneal fibroblasts. TGFβ-mediatedprocesses such as fibronectin assembly and cell migration wereinhibited. The siRNAs, when introduced subconjunctivally into mouseeyes, were also efficacious in reducing the inflammatory response andmatrix deposition. These findings indicate that siRNAs can besuccessfully delivered both in vitro and in vivo to regulate the TGFβtype II receptor level and modulate wound response. The RNA interferencetechnology may have a wide-ranging application, extending from the eyeto other organs and tissues throughout the body.

In addition to wound healing, TGFβ is known to play an important role inthe regulation of growth and differentation of many cell types. As TGFβis also known to control the accumulation of matrix proteins such ascollagen, fibronectin, thrombospondin, osteopotin, proteoglycans andglycosamineoglycans, it is thought to contribute to carcinogenic changeswithin many organ systems. Therefore, suppression of TGFβRII geneexpression may be a method of treating fibroproliferative disorders, andconnective tissue disorders.

TGFβ is also known to induce endothelial tube formation in vitro and isthought to affect the organizational process of capillary tube thatformation in vivo. TGFβ levels are known to be elevated in some cancerssuch as prostate cancer, ovarian cancer, plasmacytoma and gliablastoma.Furthermore, it is associated with angiogenesis in part by itsassociation with CTGF. Thus, suppression of TGFβRII receptor geneexpression may be a method of treating these and other types of cancers,as well as abnormal blood vessel growth.

TGFβ is also known to inhibit the growth to both T- and B-lymphocytes,natural killer cells and lymphokine-activated killer cells. Therefore,in addition to cancers, suppression of TGFβRII gene expression may be amethod of treating immune disorders such as AIDS, other viral infectionsand trypanosomal infections.

In addition, siRNA-based methods may be used to treat wounds other thanthose induce by ocular trauma, disorders or surgery. Surgical incisionsin general, trauma-induced lacerations, fibrosis due to radiationtherapy and wounds involving the peritoneum may be treated. Scarringresulting from restenosis of blood vessels, hypertrophic scars andkeloids may be treated with siRNA methods.

An ocular fibrotic wound healing response represents a significantpathophysiological issue especially as a consequence of the surgicaltreatment for glaucoma. (Migdal et al. Ophthalmology 101, 1651-1656(1994), Addicks et al., Arch. Ophthalmol. 101, 795-798 (1983)) Excessivepost-operative scarring often leads to failure of the filtrationsurgery. While the use of antimetabolites such as mitomycin-C and5-fluorouracil as conjunctival anti-scarring treatments have benefited anumber of patients, these agents are associated with potentiallyblinding complications, such as hypotony maclopathy and infection. (Khawet al., Arch. Ophthalmol. 111, 263-267 (1993), Cordeiro et al., Invest.Ophthalmol. Vis. Sci. 40, 1975-1982 (1999), Crowston et al., Invest.Ophthalmol. Vis. Sci. 39, 449-454 (1998), Stamper, Am. J. Ophthalmol.114, 544-553 (1992)).

Sequestering of mature TGFβ has been a primary target for thedevelopment of antifibrotic approaches. Antibodies to TGFβ2 have beendemonstrated to significantly reduce conjunctival scarring activity.(Cordeior et al., Invest. Ophthalmol. Vis. Sci. 40, 2225-2234 (1999),(Mead et al., Invest. Ophthalmol. Vis. Sci. 44, 3394-3401 (2003)) Inaddition, modulation of wound healing is observed when antisenseoligonucleotides (Cordeior, et al., Gene Therapy 10, 59-70 (2003), Shenet al., Eur. J. Bioichem. 268, 2331-2337 (2001)) or ribozymes (Su et al.Biochem. Biophys. Res. Commun. 278, 401-407 (2000), Yamamoto et al.,Circulation 102, 1308-1314 (2000)) to TGFβ are applied to animal modelsor cultured cells. Nevertheless, neutralizing antibodies in generalexhibit relatively weak effects as these antibodies may not gain fullaccess to the targeted molecule. (Shen et al., Eur. J. Bioichem. 268,2331-2337 (2001)). Antisense phosphorothioate oligonucleotides andribozymes can be effective, but their stability and specificity are attimes still in question. The concentration needed is also generally inthe μM range. By comparison, the siRNAs are efficacious at 200 nM andare highly specific. Therefore, the present invention specificallycontemplates compositions comprising siRNAs at a concentration of 100nM, 110 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180nM, 190 nM, 200 nM, 210 nM, 220 nM, 240 nM, 250 nM, 260 nM, 270 nM, 280nM, 290 nM, and 300 nM or more.

Such compositions of the invention will be used in methods of treatingor preventing glaucoma. In addition, recent studies have shown that TGFβmay actually be involved in the pathogenesis of primary open angleglaucoma (Inatani et al., Graefes Archive for Clinical & ExperimentalOphthalmology. 239(2):109-13, 2001; Ochiai et al., Japanese Journal ofOphthalmology. 46(3):249-53, 2002; Gattanka et al., Invest OphthalmolVis Sci., 45(1):153-8, 2004;). Down regulation of the TGFβ receptors inthe anterior chamber using siRNA against the TGFβ receptor will beanother treatment modality against the actual development or progressionof glaucoma. Therefore, the siRNA compositions of the present inventionmay be used to in treatment methods for glaucoma that has alreadydeveloped or alternatively may be used prophylactically to preventglaucoma. Those of skill in the art are aware of animal models forophthalmologic function and methods and routes of administeringtherapeutic compositions (e.g., shunts, perfusion, etc.) for thetreatment or prevention of glaucoma, see for example, Inatani et al.,supra, and Ochiai et al., supra, U.S. Pat. Nos. 6,713,498; 6,699,211;6,699,210; 6,649,625; 6,595,945; 6,531,128; 6,482,854. Each of thesedocuments are incorporated herein by reference in their entirety.

Furthermore, the use of siRNA against TGFβ receptors will be of value inpreventing restenosis of coronary vessels as well as helping to arrestthe progression of pulmonary fibrosis and pulmonary scarring fromchronic pulmonary obstructive disease as well as renal fibrosis andpostoperative scarring in the abdomen and elsewhere in the body. Thus,it is contemplated that the siRNA-based compositions of the inventionwill be useful as or in conjunction with therapeutic methods for theimprovement of circulation and hemostasis in stenotic vessels. Thus,these siRAN compositions may be used alone or in combination with (e.g.,during, before or after) by-pass surgery and revascularizationprocedures (e.g., balloon angioplasty, atherectomy, rotorary ablation(rotoblation)) which serve to improve blood flow by reducing or removingthe stenosis. These methods will be useful in reducing the thickness orpresence of neointima within the vessel wall which reduces the luminalarea of the vessel (i.e., restenosis). For further details of methodsand compositions for treating restenosis and stenosis see e.g., U.S.Pat. Nos. 6,663,863; 6,648,881; 6,596,698; 6,520,957; 6,519,488;6,458,590; 6,491,720; 6,241,718. Each of these documents areincorporated herein by reference in their entirety. These patents arelisted to show exemplary teachings in the art for the preparation ofstents and medicaments for the treatment of restenosis. The compositionsdescribed herein may be used in like manner to the medicaments describedtherein and also may be used to supplement the treatment methodsdescribed in those exemplary patents.

RNA Interference (RNAi) Technology

Variations on RNA interference (RNAi) technology is revolutionizing manyapproaches to experimental biology, complementing traditional genetictechnologies, mimicking the effects of mutations in both cell culturesand in living animals. (McManus & Sharp, Nat. Rev. Genet. 3, 737-747(2002)) The present invention demonstrates that the RNAi technology canbe successfully used to regulate wound healing response by targeting theTGFβII receptor gene. The effect is specific and potent. This technologymay be applied not only to the conjunctiva, cornea, retina and choroidof the eye, but also in other tissues throughout the body to modulatewound responses in disorders including vascular diseases, hypertensionand atherosclerosis. (Yamamoto et al., Circulation 102, 1308-1314(2000))

In the current study, RNAi was used to target the TGFβ pathway. RNAi,known to occur in animals and eukaryotes, is a process in which doublestranded RNA (dsRNA; typically >200 nucleotides in length) triggers thedestruction of mRNAs sharing the same sequence. RNAi is initiated by theconversion of dsRNA into 21-23 nucleotide fragments and these smallinterfering RNAs (siRNAs) direct the degradation of target RNAs.(Elbashir et al., Nature 411, 494-498 (2001), Fire et al., Nature 391,199-213 (1998), Hannon, G. J., Nature 418,244-251 (2002)). It has beenrapidly adopted to use for silencing genes in a variety of biologicalsystems. (Reich et al., Mol. Vis. 9, 210-216 (2003), Song et al., Nat.Med. 9, 347-351 (2003))

RNAi technology may be carried out in mammalian cells by transfection ofsiRNA molecules. The siRNA molecules may be chemically synthesized,generated by in vitro transcription, or expressed by a vector or PCRproduct. Commercial providers such as Ambion Inc. (Austin, Tex.),Darmacon Inc. (Lafayette, Colo.), InvivoGen (San Diego, Calif.), andMolecula Research Laboratories, LLC (Herndon, VA) generate custom siRNAmolecules. In addition, commercial kits are available to produce customsiRNA molecules, such as SILENCER™ siRNA Construction Kit (Ambion Inc.,Austin, Tex.) or psiRNA System (InvivoGen, San Diego, Calif.). ThesesiRNA molecules may be introduced into cells through transienttransfection or by introduction of expression vectors that continuallyexpress the siRNA in transient or stably transfected mammalian cells.Transfection may be accomplished by well known methods including methodssuch as infection, calcium chloride, electroporation, microinjection,lipofection or the DEAE-dextran method or other known techniques. Thesetechniques are well known to those of skill in the art.

The siRNA molecules may be introduced into a cell in vivo by localinjection of or by other appropriate viral or non-viral deliveryvectors. Hefti, Neurobiology, 25:1418-1435 (1994). For example, thesiRNA molecule may be contained in an adeno-associated virus (AAV)vector for delivery to the targeted cells (e.g., Johnson, InternationalPublication No. WO95/34670; International Application No.PCT/US95/07178). The recombinant AAV genome typically contains AAVinverted terminal repeats flanking the siRNA sequence operably linked tofunctional promoter and polyadenylation sequences. Alternative suitableviral vectors include, but are not limited to, retrovirus, adenovirus,herpes simplex virus, lentivirus, hepatitis virus, parvovirus,papovavirus, poxvirus, alphavirus, coronavirus, rhabdovirus,paramyxovirus, and papilloma virus vectors.

Nonviral delivery methods include, but are not limited to,liposome-mediated transfer, naked DNA delivery (direct injection),receptor-mediated transfer (ligand-DNA complex), electroporation,calcium phosphate precipitation, and microparticle bombardment (e.g.,gene gun). Methods of introducing the siRNA molecules may also includethe use of inducible promoters, tissue-specific enhancer-promoters, DNAsequences designed for site-specific integration, DNA sequences capableof providing a selective advantage over the parent cell, labels toidentify transformed cells, negative selection systems and expressioncontrol systems (safety measures), cell-specific binding agents (forcell targeting), cell-specific internalization factors, andtranscription factors to enhance expression by a vector as well asmethods of vector manufacture.

The preferred siRNA molecule is 19-25 base pairs in length, mostpreferably 21-23 base pairs, and is complementary to the target genesequence. The siRNA molecule preferably has two adenines at its 5′ end,but may not be an absolute requirement. The siRNA sequences that contain30-50% guanine-cytosine content are known to be more effective thansequences with a higher guanine-cytosine content. Therefore, siRNAsequence with 30-50% are preferable, while sequences with 40-50% aremore preferable. The preferred siRNA sequence also should not containstretches of 4 or more thymidines or adenines.

The present specification provides details of studies performed withsiRNAs designed to target the TGFβ type II receptor (TGFβII) gene. Thetarget sequence selected should not be highly structured or bound byregulatory proteins. Preferably, the siRNA molecules of the inventionshould be directed to different positions within the target genesequences. For example, siRNA target sequences NK1, NK2, SS1 and SS2(SEQ ID NO: 1-4) are directed to different portions of the TGFβRII gene.In particular nucleotides NK1 spans nucleotides 529-612, NK2 spansnucleotides 1113-1133, SS1 spans nucleotides 1253-1273 and SS2 spansnucleotides 948-969 of the TGFβRII gene. Additional siRNA targetsequences that may be effective for suppressing TGFβRII gene expressionare set out in Table 1 below. These sequences were derived by analyzingthe human TGFβRII sequence (M85079) using the publicly available siRNATarget Finder program at the Ambion, Inc. web site. The sequences werescreened by BLAST searching the Genbank database for homologoussequences. Any sequence containing more than 16 nucleotides match to anon-TGFβRII sequence were eliminated from further consideration.

Sequences with a GC content between 30-50% were further analyzed. Thosesequences containing four consecutive A, C, G or T bases wereeliminated. This analysis identified an additional 49 siRNA moleculesthat are contemplated to be effective in inhibiting TGFβRII gene. Thesesequences are shown in FIG. 3. The siRNA molecules that contain up to 2mismatches are effective in inhibiting TGFβRII expression. Theeffectiveness of the siRNA containing mismatches may be dependent ontheir position in the sequence. Thus, it is likely that other siRNAsequences may be derived from the 4 already tested (NK1, NK2, SS1 andSS2) and those indicated in FIG. 3.

The present specification provides details of studies performed withsiRNAs designed to target the TGFβ type II receptor (TGFβRII) gene. Incultured human corneal fibroblasts, the siRNAs effectively suppressedgene expression of the receptor, reduced TGFβ-mediated matrix depositionand retarded cell migration. In addition, the data presented hereinshows in an in vivo model that siRNAs specific for TGFβRII can reduceinflammation and regulate wound repair in the conjunctiva of mouse eyes.The siRNA molecules of the present invention also effectively suppressTGFβRII gene expression in human umbilical vein endothelial cells.

siRNAs specific to human TGFβRII can inhibit the receptor expression incultured human corneal fibroblasts as shown by immunofluorescence,Western blotting and real time PCR analyses. Four concentrations ofsiRNAs ranging from 25 to 200 nM and four time points from 16 to 72hours were tested. The inhibitory response is both dose and timedependent. Specificity of the siRNAs for the TGFβRII has also beenestablished. All four siRNAs tested were found to be efficacious,although two of them showed greater effect. Given the teachings providedherein, one of skill in the art would expect that other siRNAs deducedfrom the cDNA sequence of human TGFβRII also will be as effective.

Assays to Test Efficacy of siRNA Specific to Human TGFβ Type II ReceptorIn Vitro Models

Corneal fibroblasts constitutively express TGFβ. (Song et al., J. Cell.Biochem. 77, 186-199 (2000), Imanishi et al., Prog. Retin. Eye Res. 19,113-129 (2000)) The effects of siRNAs in blocking autocrine TGFβsignaling in corneal fibroblasts was examined and are described herein.The functional roles of the siRNAs are thus well established in this invitro culture model.

TGFβ has been shown to enhance the expression of matrix molecules suchas fibronectin and collagen type I (Song et al., J. Cell. Biochem. 77,186-199 (2000), Massague, Annu. Rev. Cell Biol. 6, 597-641 (1990)) andto facilitate migration of corneal fibroblasts, (Imanishi et al., Prog.Retin. Eye Res. 19, 113-129 (2000), Andersen et al, Curr. Eye Res. 16,605-613 (1997)), and the steps involved in the complex wound repairprocess. (Clark, Physiology, Biochemistry and Molecular Biology of theSkin, Oxford University Press. P. 576-601, 1997) As has beendemonstrated in hepatic stellate cells with antisense RNA complementaryto TGFβ1, (Arias et al., Cell Growth Differ. 13, 265-273 (2002))diminished receptor level and blockade of receptor binding for TGFβcaused a reduction in the secreted fibronectin level and itsincorporation into the matrix. Corneal fibroblast migration is alsomarkedly retarded.

Given the teachings of the present invention, those of skill in the artare instructed to produce siRNA molecules discussed herein and employsuch molecules in in vitro assays to assess the effects of such siRNAmolecules on migration of corneal fibroblasts, the expression offibronectin, and/or the expression of collagen type I. Any decrease ordiminution of the level of migration of corneal fibroblasts, the levelof expression and/or secretion of either fibronectin or collagen type Iwill be indicative of the given siRNA molecule being effective for useas a therapeutic agent in accordance with the present invention.

Mouse Models

The therapeutic effects of the TGFβ specific siRNA molecules are alsodemonstrated in a conjunctival scarring mouse model. The model wassimilar to that described previously by Reichel et al. (Br. J.Ophthalmol. 82, 1072-1077 (1998)). However, instead of injecting onlyPBS into the subconjunctival space, the injected PBS was mixed withlatex beads to have an improved mouse model with augmented inflammatoryand scarring response. siRNA at 200 nM clearly showed its effectivenessin reducing the inflammatory and fibrotic response in this new mousemodel. Those of skill in the art could repeat these model studies withany other TGFβ specific siRNA molecule. Any other molecule that reducesthe inflammatory or fibrotic response in this mouse model iscontemplated to be a useful siRNA molecule of the invention.

Cell Growth Assays

TGFβ is known to stimulate fibroblast proliferation and inhibitproliferation of epithelial cells, in particular tumor cells. Therefore,measuring the effect of siRNA on TGFβ-induced fibroblast proliferationor epithelial cell growth inhibition is a method for evaluating theeffectiveness of the siRNA molecules.

Cell growth may be monitored by measuring DNA synthesis. DNA synthesismay be measured using [³H]-thymidine incorporation in cells as describedin Lee et al., (Endocrinology 136:796-803, (1995)). Cells are seeded atapproximately 2×10⁴ per well (24-well plate) and are incubated for 22hours in 1 ml culture medium with or without 1% PBS and containing TGFβat selected concentrations. Then 2 mCi per well [³H]-thymidine is added,subsequently incubation continues for 4 hours, and radioactivity iscounted with a scintillation counter.

Cell proliferation can be measured by cell counting. Cells are seeded(24-well plates) in culture medium with or without 1% FBS and medium ischanged every other day. At the end of a 4-day culture, cells aretrypsinized and counted in a Coulter counter.

TGFβRII Activation Assays

The use of the p3TP-lux construct allows for evaluation of activation ofthe TGFβ type II receptor. Cells are seeded at 1×10⁵ cells per well in6-well plates and are transiently transfected with the plasmid p3TP-Luxusing lipofection according to manufacturer's instructions (Lifetechnologies, Gaithersburg, Md.). p3TP-Lux contains three12-O-tetradecanoylphorbol-13-acetate-responsive elements from the humancollagen gene and one TGFβ-responsive element from the human plasminogenactivator inhibitor-1 (PAI-1) promoter linked to the luciferase reportergene (Wrana et al., Cell 71: 1003-14, (1996)). Cells are incubated with1 μg/ml p3TP-Lux and 12 μg/ml Lipofectamine for 24 hours. Subsequently,cells are treated with 5 ng/ml TGFβ in RPMI for 24 hours and lysed withextraction buffer (100 mM potassium phosphate, pH 7.5, 1% Triton X-100,100 mg/ml bovine serum albumin, 2.5 mM phenylmethylsulfonylfluoride, 1mM dithiothreitol). Lysates are diluted into reaction buffer (75 mMMgCl₂, 1 M glycylglycine, pH 7.8, 100 mg/ml bovine serum albumin, 60mg/ml ATP) and are assayed for luciferase activity using a luminometer.

Use of this assay allows one to evaluate the effectiveness of the siRNAon TGFβRII activity. An effective siRNA molecule of the presentinvention will inhibit the amount of signaling through the TGFβRIIreceptor as it will reduce the number of receptors available forsignaling. Preferably, the effective siRNA molecule will inhibitsignaling through TGFβRII by at least 20%, or more preferably by atleast 25%, 30%, 35%, 40% or 45%. It is highly preferable that theeffective siRNA molecule inhibit signaling through the TGFβRII by atleast 50%, 55%, 60%, 65%, 70, 75% or more.

Chemotaxis Assays.

TGFβ is a cytokine and those of skill in the art monitor the activity ofsuch agents through well known chemotaxis assays. Exemplary chemotaxisassays that may be performed are described in Martinet et al., J.Immunol. Meth., 174:209, 1994 and Keller et al., J. Immunol. Meth.,1:165, 1972. Briefly, 20 ml of peripheral blood is collected from healthvolunteers in 10 ml heparinized tubes. Blood is diluted 1:1 and thenunder laid with 10 ml of Histopaque (Sigma). After centrifugation at 400g for 25 minutes, cells at the interface are collected and washed twicein PBS. Cells are resuspended in DMEM (Life Technologies, Gaithersburg,Md.) with 100 U/ml penicillin and 100 μg/ml streptomycin (tissue cultureantibiotics, Life Technologies) at 106/ml. Sterile bovine serum albumin(Sigma) is added to final concentration of 0.2 mg/ml.

100 μl of this cell suspension is added to each transwell insert(Costar). DMEM with antibiotics and 0.2% BSA with or without siRNAmolecules is added to the lower wells in the 24 well plate. Transwellinserts are placed into the lower walls, and incubated at 37N C for 90minutes. At the completion of the incubation period inserts are removedand the adherent cells are removed. The entire insert is then stainedwith Wright-Giemsa. Cells adherent to the lower surface of the insertand those that migrated to the lower well are counted under microscope,and added together to obtain a total number of migrating cells.

Assay of Chemoattractant and Cell-Activation Properties.

The effects of siRNA directed to TGFβRII upon humanmonocytes/macrophages or human neutrophils may be evaluated, e.g., bymethods described by Devi et al., J. Immunol., 153:5376-5383 (1995) forevaluating murine TCA3-induced activation of neutrophils andmacrophages. Indices of activation measured in such studies includeincreased adhesion to fibrinogen due to integrin activation, chemotaxis,induction of reactive nitrogen intermediates, respiratory burst(superoxide and hydrogen peroxide production), and exocytosis oflysozyme and elastase in the presence of cytochalasin B.

As discussed by Devi et al., these activities correlate to severalstages of the leukocyte response to inflammation. This leukocyteresponse, reviewed by Springer, Cell, 76:301-314 (1994), involvesadherence of leukocytes to endothelial cells of blood vessels, migrationthrough the endothelial layer, chemotaxis toward a source of chemokines,and site-specific release of inflammatory mediators.

Assays of Effect on Myeloid Progenitor Cells.

The inhibition of TGFβ-induced suppression of hematopoiesis may betested in assays of stem/progenitor cell function and number, includingLTC-IC, CFU-GEMM, CFU-GM, BFU-E. These assays are well known to those ofskill in the art and are relatively straightforward to set up asdescribed in for example Broxmeyer et al., Blood, 76:1110 (1990).Briefly, bone marrow cells are collected from human donors afterobtaining informed consent. Low density human bone marrow cells at5×104/ml are plated in 1% methylcellulose in Iscove's Modified EssentialMedium (Biowhitaker, Walkersville, Md.) supplemented with 30% FCS(Hyclone), recombinant human erythropoietin (EPO, 1 U/ml, Amgen,Thousand Oaks, Calif.), recombinant human interleukin-3 (IL-3, 100 U/ml,Immunex, Seattle, Wash.), and recombinant human stem cell factor (SCF,50 ng/ml, Amgen) for colony forming unit granulocyte/macrophage(CFU-GM), colony forming unitgranulocyte/erythrocyte/macrophage/megakaryocyte (CFU-GEMM) or blastforming unit-erythrocyte (BFU-E) analysis. Cultures are incubated at 5%CO2 and low oxygen tension (5%) for 14 days, and then scored for colonyformation using an inverted microscope in a blinded fashion.

Assays for Effects on Myeloid Cell Lines.

The effect of siRNA on TGFβ-induced inhibition of myeloid cellproliferation also may be a useful test of functional activity of thesiRNA molecules. Such a functional assay may be assessed using the humanmyeloid cell lines TF-1 and MO7E (Avanzi et al., Brit. J Haematol.,69:359; 1988), which require GM-CSF and SCF for maximal proliferation.The cytokine-dependent primitive acute myeloid leukemia cell lines TF-1and MO7E may be cultured in RPMI 1640 (Life Technologies, Gaithersburg,Md.) plus 10% FCS (Hyclone) and 100 U/ml penicillin and 100 μg/mlstreptomycin (tissue culture antibiotics, Life Technologies,Gaithersburg, Md.). This media is supplemented withgranulocyte-macrophage colony stimulating factor (GM-CSF, 100 U/ml,Immunex, Seattle, Wash.) and stem cell factor (SCF, 50 ng/ml, Amgen,Thousand Oaks, Calif.) to promote normal log phase growth.

Assays for Effect on Chronic Myelogenous Leukemia Progenitors.

The effect of siRNA on TGFβ-induced inhibition of progenitorproliferation in chronic myelogenous leukemia (CML) may be evaluatedusing colony formation assays as described in Hromas et al., Blood,89:3315-3322 (1997). Briefly, bone marrow cells are collected from sixCML patients in chronic phase. Low density marrow cells at for example,5×10⁴ cells/mL are plated in 1% methylcellulose in Iscove's modifiedDulbecco's medium supplemented with 30% fetal calf serum, 1 U/mL humanerythropoietin. (Epogen®, Amgen), 100 U/mL human interleukin-3 (GeneticsInstitute) and 50 ng/mL human stem cell factor (Amgen), in the presenceor absence of an appropriate concentration of TGFβ (e.g. 100 ng/ml)alone or in combination with other chemokines such as EXODUS, MIP-1α andthe like.

Cultures are incubated at 5% CO₂ and low (5%) oxygen tension for 14days, and then scored using an inverted microscope for CFU-GM, CFU-GEMMand BFU-E. Colony counts for cultures treated with chemokines arecompared to colony counts of the control cultures and were expressed asa percentage of control CFU or FU.

As stated earlier, the assays described above are intended to exemplifythe types of assays that may be conducted to determine the in vitro andin vivo effects of the siRNA molecules of the present invention. Theseare by no means the only assays known to be used for determine TGFβactivity. Those of skill in the art will know of other assays that maybe substituted for these described above but nonetheless measure similarparameters of function and activity.

Angiogenesis Assays

The effect of siRNA molecules on angiogenesis may be monitored using thefollowing assays. Angiogenesis is the multistep process of new capillaryformation originating from sprouting of endothelial cells from the wallof an existing small blood vessel. In order for new capillary tubes toform, endothelial cells must elongate and migrate.

A tube formation assay may be utilized to determine if the siRNAmolecules targeting TGFβRII inhibit tube formation in endothelial cellssuch as HUVEC cells. For example endothelial tube formation assays maybe carried out in vitro using Matrigel. When endothelial cells areplated on BD Matrigel™ (BD Biosciences), the cells stop proliferating,and display high motility and cell-cell communication. Furthermore,within 24 hours, the cells align and form a three-dimensional network ofcapillary tubes that has been proposed as a model of endothelial celldifferentiation as well as one of the final steps of the angiogeniccascade.

A 24-well tissue culture plate is coated with 500 μl of the MatrigelMatrix with reduced growth factors and allowed to gel thoroughly byincubating at 37° C. for at least 30 minutes. After the Matrigel forms agel, endothelial cells such as bovine aortic endothelial cells (BAEC) orhuman umbilical vein endothelial cells (HUVEC), are washed and seeded onMatrigel coated wells. The cells are treated with TGFβ in the presenceand absence of siRNA molecules targeted to TGFβRII, To view tubeformation, cells are treated with 1 mM Calcein AM (Molecular Probes)diluted at 1:2000 in media, incubated in the dark for at least 15minutes, and subsequently washed with media+10% FBS.

Other assays to evaluate the effect of siRNA molecules on TGFβ-inducedangiogeneis include endothelial cell proliferation assays andendothelial cell migration assays. In addition, alterations inendothelial cells occur during angiogenesis as vessels invade tumors,and have effects on endothelial cell morphology and function.Endothelial cell morphology may be evaluated using immunohistochemistryor electron microscopy to view endothelial cell sprouting, migration,and proliferation.

The Chicken Chorioallantoic Membrane (CAM) assay is also a well knownmethod of evaluating angiogenesis. The developing chicken embryo issurrounded by a chorioallantoicmembrane, which becomes vascularized asthe embryo develops. Tissue grafts are placed on the CAM through awindow made in the eggshell. This causes a typical radial rearrangementof vessels towards, and a clear increase of vessels around the graftwithin four days after implantation. Blood vessels entering the graftare counted under a stereomicroscope. To assess the anti-angiogenic orangiogenic activity of the siRNA molecules, the compounds are eitherprepared in slow release polymer pellets, absorbed by gelatin sponges orair-dried on plastic discs and then implanted onto the CAM. In the CAMassay, siRNA of the present invention that lead to the regression ofnewly developed CAM vasculature are determined to be effectiveinhibitors of TGFβ-induced angiogenesis.

The effect of the siRNA molecules of the present invention onTGFβ-induced angiogeneis may also be measured in the mouse cornea usingthe micropocket assay. The mouse cornea presents an in vivo avascularsite. This makes it a very good model for studying angiogenesis, as thegrowth of new blood vessels easily can be studied under microscope. Anyvessels penetrating from the limbus into the corneal stroma can beidentified as newly formed. To induce an angiogenic response, slowrelease polymer pellets (i.e. poly-2-hydroxyethyl-methacrylate (hydron)or ethylene-vinyl acetate copolymer (ELVAX)), containing an TGFβ isimplanted in “pockets” created in the corneal stroma of a mouse. After4-6 days, new vessel growth occurs. The vascular response can bequantified by computer image analysis after perfusion of the cornea withIndia ink. The blood vessels in this model can also be studiedultrastructurally by electron microscope, or by the use ofimmunohistochemistry.

Pharmaceutical Compositions.

Where clinical applications are contemplated, it will be necessary toprepare the viral expression vectors, nucleic acids and othercompositions identified by the present invention as pharmaceuticalcompositions, i.e., in a form appropriate for in vivo applications.Generally, this will entail preparing compositions that are essentiallyfree of pyrogens, as well as other impurities that could be harmful tohumans or animals. In preferred embodiments, the present inventioncontemplates pharmaceutical compositions containing siRNA moleculesdescribed as the present invention.

The active compositions of the present invention include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. The pharmaceuticalcompositions may be introduced into the subject by any conventionalmethod, e.g., by intravenous, intradermal, intramusclar, intramammary,intraperitoneal, intrathecal, intraocular, retrobulbar, intrapulmonary(e.g., term release); by oral, sublingual, nasal, anal, vaginal, ortransdermal delivery, or by surgical implantation at a particular site,e.g., embedded under the splenic capsule, brain, or in the cornea. Thetreatment may consist of a single dose or a plurality of doses over aperiod of time.

The active compounds may be prepared for administration as solutions offree base or pharmacologically acceptable salts in water suitably mixedwith a surfactant, such as hydroxypropylcellulose. Dispersions also canbe prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like that do notproduce adverse, allergic, or other untoward reactions when administeredto an animal or human. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients also can be incorporatedinto the compositions.

For oral administration the polypeptides of the present invention may beincorporated with excipients and used in the form of non-ingestiblemouthwashes and dentifrices. A mouthwash may be prepared incorporatingthe active ingredient in the required amount in an appropriate solvent,such as a sodium borate solution (Dobell's Solution). Alternatively, theactive ingredient may be incorporated into an antiseptic wash containingsodium borate, glycerin and potassium bicarbonate. The active ingredientmay also be dispersed in dentifrices, including: gels, pastes, powdersand slurries. The active ingredient may be added in a therapeuticallyeffective amount to a paste dentifrice that may include water, binders,abrasives, flavoring agents, foaming agents, and humectants.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups alsocan be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups alsocan be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration.

“Unit dose” is defined as a discrete amount of a therapeutic compositiondispersed in a suitable carrier. For example, where siRNA molecules arebeing administered parenterally, siRNA compositions are generallyinjected in doses ranging from 1 mg/kg to 100 mg/kg body weight/day,preferably at doses ranging from 0.1 mg/kg to about 50 mg/kg bodyweight/day. Parenteral administration may be carried out with an initialbolus followed by continuous infusion to maintain therapeuticcirculating levels of drug product. Those of ordinary skill in the artwill readily optimize effective dosages and administration regimens asdetermined by good medical practice and the clinical condition of theindividual patient.

The frequency of dosing will depend on the pharmacokinetic parameters ofthe agents and the routes of administration. The optimal pharmaceuticalformulation will be determined by one of skill in the art depending onthe route of administration and the desired dosage. See for exampleRemington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publ. Co,Easton Pa. 18042) pp 1435-1712, incorporated herein by reference. Suchformulations may influence the physical state, stability, rate of invivo release and rate of in vivo clearance of the administered agents.Depending on the route of administration, a suitable dose may becalculated according to body weight, body surface areas or organ size.Further refinement of the calculations necessary to determine theappropriate treatment dose is routinely made by those of ordinary skillin the art without undue experimentation, especially in light of thedosage information and assays disclosed herein as well as thepharmacokinetic data observed in animals or human clinical trials.

Appropriate dosages may be ascertained through the use of establishedassays for determining blood levels in conjunction with relevantdose-response data. The final dosage regimen will be determined by theattending physician, considering factors which modify the action ofdrugs, e.g., the drug's specific activity, severity of the damage andthe responsiveness of the patient, the age, condition, body weight, sexand diet of the patient, the severity of any infection, time ofadministration and other clinical factors. As studies are conducted,further information will emerge regarding appropriate dosage levels andduration of treatment for specific diseases and conditions.

In gene therapy embodiments employing viral delivery, the unit dose maybe calculated in terms of the dose of viral particles beingadministered. Viral doses include a particular number of virus particlesor plaque forming units (pfu). For embodiments involving adenovirus,particular unit doses include 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰,10¹¹, 10¹², 10¹³ or 10¹⁴ pfu. Particle doses may be somewhat higher (10to 100-fold) due to the presence of infection defective particles.

It will be appreciated that the pharmaceutical compositions andtreatment methods of the invention may be useful in fields of humanmedicine and veterinary medicine. Thus the subject to be treated may bea mammal, preferably human or other animal. For veterinary purposes,subjects include for example, farm animals including cows, sheep, pigs,horses and goats, companion animals such as dogs and cats, exotic and/orzoo animals, laboratory animals including mice rats, rabbits, guineapigs and hamsters; and poultry such as chickens, turkey ducks and geese.

Combined Therapy.

In addition to therapies based solely on the delivery of siRNA moleculesand related composition, combination therapy is specificallycontemplated. In the context of the present invention, it iscontemplated that siRNA methods could be used similarly in conjunctionwith other agents for promoting wound-healing, reducing scarring,inhibiting angiogenesis, or those used in the therapy of the disordersenumerated herein. It is also contemplated that the siRNA moleculesdirected to TGFβRII could be used in conjunction with other siRNAmolecules that promote wound healing, reducing scarring, inhibitingangiogenesis or those used in the therapy of the disorders describedherein.

To achieve the appropriate therapeutic outcome, be it a decrease inscarring, decrease in fibrogen accumulation, reduction in angiogenesisor any other use for the siRNA molecules discussed herein, using themethods and compositions of the present invention, one would generallycontact a “target” cell with a siRNA expression construct and at leastone other therapeutic agent (second therapeutic agent). Thesecompositions would be provided in a combined amount effective to producethe desired therapeutic outcome. This process may involve contacting thecells with the expression construct and the agent(s) or factor(s) at thesame time. This may be achieved by contacting the cell with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes the expressionconstruct and the other includes the second therapeutic agent.

Alternatively, the siRNA treatment may precede or follow the other agenttreatment by intervals ranging from minutes to weeks. In embodimentswhere the second therapeutic agent and expression construct are appliedseparately to the cell, one would generally ensure that a significantperiod of time did not expire between the time of each delivery, suchthat the agent and expression construct would still be able to exert anadvantageously combined effect on the cell. In such instances, it iscontemplated that one would contact the cell with both modalities withinabout 12-24 hours of each other and, more preferably, within about 6-12hours of each other, with a delay time of only about 12 hours being mostpreferred. In some situations, it may be desirable to extend the timeperiod for treatment significantly, however, where several days (2, 3,4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse betweenthe respective administrations.

Local delivery of siRNA expression constructs or sequences to patientsmay be a very efficient method for delivering the siRNA molecules tocounteract a clinical disease. Similarly, the second therapeutic agentmay be directed to a particular, affected region of the subject's body.Alternatively, systemic delivery of expression construct and/or thesecond therapeutic agent may be appropriate in certain circumstances.

Other antiproliferative and anti-angiogenic compositions which may beeffective include in combination treatments with the siRNA molecules ofthe present invention include anti-cancer drugs mitomycin-C and5-fluorouracil, agaricus bisporus lectin, metallocomplexes such aszinc-desferrioxaminde or gallium-desferrioxamine, methyl xanthinederivatives such as pentoxifylline, collagen-based sealants such as GEAmidon Oxyde. In addition, agents that inhibit VEGF, fibroblast growthfactors, connective tissue growth factors and matrix metalloproteinaseinhibitors such as ilomastat are contemplated as second therapeuticagents for use with the siRNA molecules of the present invention. Suchinhibitors include siRNA molecules that target VEGF, fibroblast growthfactors, connective tissue growth factors or the respective receptorsfor these growth factors and matrix metalloproteinases.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Materials & Methods

Human Corneal Fibroblast Cultures.

Normal human corneas from donors aged 13, 29, 34, 45, and 47 years wereobtained from either the Illinois Eye Bank (Chicago, Ill.) or theNational Disease Research Interchange Philadelphia, Pa.). Theprocurement of tissue was approved by the IRB Committee at theUniversity of Illinois at Chicago in compliance with the declaration ofHelsinki. The endothelial and epithelial layers were removed from thecorneas and the stroma was used as explants to initiate cornealfibroblast cultures. The cells were maintained in Dulbecco's modifiedEagle's minimum essential medium (MEM) supplemented with glutamine, 10%fetal calf serum, 5% calf serum, nonessential and essential amino acidsand antibiotics as previously described in Yue and Blum. (Vision Res.21, 41-43 (1981)) Third- to fifth-passaged cells were used for thestudy.

TGFβII Receptor siRNA Sequences.

Four sequences for the TGFβII receptor siRNA were derived from the humanTGFβII receptor sequence (Genbank Accession Number: M85079). The siRNAswere custom synthesized and purified by Dharmacon Research (Lafayette,Colo.). The target sequences (5′ to 3′) were as follows, with theposition of the first nucleotide in the human TGFβII receptor sequenceshown in brackets: NK1:  (529) AATCCTGCATGAGCAACTGCA (SEQ ID NO: 1) NK2:(1113) AAGGCCAAGCTGAAGCAGAAC (SEQ ID NO: 2) SS1: (1253)AAGCATGAGAACATACTCCAG (SEQ ID NO: 3) SS2:  (948) AAGACGCGGAAGCTCATGGAG(SEQ ID NO: 4)

RNA of a scrambled sequence was used as a control.

Transfection of siRNA Duplexes

Normal human corneal fibroblasts were plated at 50-70% confluence ontoLab-Tek 4- or 8-well chamber slides, coverslips, or 6-well plates theday prior to the transfection. Transfection complexes were prepared byadding 2 μl of TransIT-TKO reagent (Takara Mirus Corporation, Madison,Wis.) to 50 μl of serum-free media, vortexing and incubating the mixtureat room temperature for 10 min. To the mixture, anti-TGFβII receptorsiRNA duplex (25, 50, 100, or 200 nM final concentration) was added. Thesolution was further mixed by gently pipeting and was incubated foranother 20 minutes. The final mixture was then added dropwise to thecells in complete media. After gentle rocking, the cells were incubatedat 37° C. for 16, 24, 48, or 72 hours before assaying for geneexpression. As controls, corneal fibroblasts were either untreated ortreated only with the transfection reagent. Non-specific scrambled siRNAduplex (Dharmacon; 100 and 200 mM) was also used in place of the TGFβRIIspecific siRNAs.

Immunofluorescence.

At selected time points after siRNA transfection, cells in coverslips or8-well chamber slides (Nalge Nunc International, Naperville, Ill.) werefixed with 2% formaldehyde solution and permeabilized with 0.1%Triton-X100 in PBS. Cells were blocked for 45 minutes at roomtemperature in 10% heat-inactivated normal goat serum (Colorado SerumCompany, Denver, Colo.), and incubated with a rabbit anti-TGFβIIreceptor antibody (1:100, Santa Cruz Biologicals, Santa Cruz, Calif.,SC1700) for 60 min. Following washes, a goat FITC-anti-rabbit (SouthernBiotechnology) at 1:200 was applied for a 60-minutes incubation. Thenuclei of the cells were counterstained with DAPI(4′,6′-diamidino-2-phenylindole dihydrochloride). The slides wereexamined by epifluorescence under a Zeiss Axiovert fluorescencemicroscope (Carl Zeiss, Jena, Germany).

For fibronectin staining, cells on Lab-Tek 4-well glass chamber slideswere fixed 48 hours after transfection in ice cold methanol.Immunofluorescence was performed using a rabbit anti-human fibronectin(1:100, BD Science, Lexington, Ky.) as the primary antibody andFITC-conjugated goat anti-rabbit IgG (1:100, Jackson ImmunoResearch,West Grove, Pa.) as the secondary antibody. The slides were mounted inVectashield (Vector Laboratories, Burlingame, Calif.) with DAPI. Thestaining was examined under a Zeiss 100M microscope.

Western Blotting.

After siRNA transfection, the media were removed and corneal fibroblastsin 6-well plates were harvested. Cells were lysed in a Triton buffer,followed by addition of sodium dodecyl sulfate (SDS) sample buffer.Protein samples were separated on a 10% SDS-polyacrylamide gel,transferred to nitrocellulose membranes and blocked with BLOTTO.Subsequently, blot was incubated with rabbit anti-TGFβII receptor at1:200 dilution (of course, other dilutions e.g., 1:2000, and dilutionsin between these figures also could be used) and horseradish peroxidase(HRP)-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch). Signalswere detected by chemiluminescence.

For fibronectin study, corneal fibroblasts after transfection for 48hours were incubated with serum-free MEM for 24 hours. The media werecollected and the cells were lysed on ice in 10 mM Tris-HCl (pH 8.0),150 mM NaCl, 0.5% NP40, 2 mM phenylmethylsulfonyl fluoride, and 1×cocktail protease inhibitors (Roche). Cellular debris was pelleted, andthe proteins in the lysate were quantified by Bradford protein assay.After adjusting the protein amounts, equal aliquots of media sampleswere resolved on 10% SDS-polyacrylamide gels under reducing conditions.The proteins were electroblotted onto nitrocellulose membranes. Afterblocking with 5% nonfat dry milk, the membranes were incubated withrabbit anti-human fibronectin (1:5000) and HRP-goat anti-rabbit IgG(1:10,000). Protein bands were detected using SuperSignal Substrate fromPierce (Rockford, Ill.). Densitometric analysis was performed to measurethe intensity of the fibronectin bands with the use of 1D Image Analysissoftware (Kodak Digital Imaging, Eastman Kodak Company, New Haven,Conn.).

Real Time PCR.

Total RNA was extracted with Trizol from cells treated for 24, 48, and72 hours with scrambled, NK1, or SS1 siRNA. Real time PCR was performedaccording to methods known to those of skill in the art.

Cell Migration Assay.

A wound scratch assay was used to assess cell migration. Forty eighthours after transfection, corneal fibroblasts in 24-well plates werescratched with a sterile P20 pipette tip as previously described inMostafavi-Pour et al., J. Cell Biol. 161: 155-167 (2003). The ability ofcells to migrate into the wound was examined under phase contrastmicroscopy 7 hours after wounding. To quantify the extent of migration,total area of the wound in each 10× field and the areas devoid of cellswithin the wound were measured with the use of the Image Processing ToolKit version 3.0 (an Adobe Photoshop 7:1 plugin software, ReindeerGraphics, Inc., Asheville, N.C.). A total of 10 fields were analyzed andthe mean percentage of areas covered by the migratory cells in eachspecimen was calculated. Student's t tests were used for statisticalevaluation. All experiments were repeated at least 3 times.

Mouse Model of Conjunctival Scarring.

All experiments were performed using 6 week old C57BL6 mice. Treatmentof the animals was conformed to the ARVO Statement for the Use ofAnimals in Ophthalmic and Vision Research. Mice underwent generalanesthesia with intraperitoneal injections (pentobarbital, 0.1 ml/10 gbody weight). Surgery was performed as reported previously withmodifications. (Reichel et al. Br. J. Ophthalmol. 82, 1072-1077 (1998))A blunt dissection of the temporal subconjunctival space was performedusing 1 ml syringe and 30 gauge needle by injecting of sterile PBS (pH7.4) containing latex beads (1.053 μm diameter, 300 μg/ml, Polysciences,Warrington, Pa.) with transfection reagent mixed with 200 nM NK1, SS1,or scrambled missense oligonucleotide. One eye of each mouse was treatedwith HK1 or SS1, and the contralateral eye was treated with thescrambled siRNA in a double masked manner. Eyes in other mice wereeither left untreated or injected with PBS and latex beads alone toserve as controls. Mice were sacrificed by cervical dislocation 2, 7,and 14 days after surgery. For each treatment/time point, three micewere used.

Eyes enucleated eyes were fixed at room temperature with 10% bufferedformalin for 24 hours, and were processed for paraffin sections.Five-μm-thick paraffin-embedded sections were deparaffinized,rehydrated, and stained with hematoxylin and eosin (H & E) to assess theinflammatory reaction and picrocirius red to demonstrate collagendeposition.

Example 2 Suppression of TGFβII Receptor Protein and mRNA Expression

Human corneal fibroblasts were transfected with all four exemplarysiRNAs designed using the TransIT-TKO reagent. The cellular uptake ofoligonucleotides was demonstrated by fluorescence microscopy using theCy3-labeled luciferase. The transfection seemed to be extremelyefficient, with more than 90% of the cells displaying red fluorescence.Little cytotoxicity of the transfection reagent or the siRNAs wasobserved.

Immunofluorescence analyses showed that TGFβRII was distributeddiffusely in the cytoplasm of untreated control corneal fibroblasts(FIG. 1, row 1). When treated with 100 nM of SS1 siRNA for 48 h, theTGFβRII staining intensity was dramatically reduced (FIG. 1, rows 3 and4). At 100 nM, NK1, NK2 and SS2 siRNAs also suppressed the TGFβRIIintensity. While not evident at the lowest concentration (25 nM) and theshortest time point (16 h) tested, the inhibiting effects, to varyingdegrees, were also observed for all four siRNAs tested with otherconcentrations (50 and 200 nM) and time points (24 and 72 h). Overall,NK1 and SS1 appeared to result in a greater inhibition than the others.Cells treated with scrambled siRNA (FIG. 1, row 2) showed a similarintensity and pattern as the untreated control cells, demonstrating thespecificity of NK1 and SS1 effects.

Western blotting (FIG. 2) yielded a 73-75 kDa band (a diffuse band asthe receptor is a glycoprotein) immunoreactive to anti-TGFβRII in thevehicle-treated control and scrambled siRNA-transfected samples. Therewas no discernible difference in the TGFβRII protein level at the 16hours time point except for the cells treated with SS1 (lane 6) where areduction was seen. At 48 h, both NK1 (lanes 9 and 10) and SS1 (lanes 11and 12) siRNAs showed a marked decrease in signal intensity for TGFβIIreceptor compared to control cells (lane 8). A densitometric analysissuggests a 70-85% reduction of the TGFβRII in the siRNA treatedimmunoblots. NK1 siRNA appeared to be more effective than SS1 inreducing the TGFβRII expression at this time point at both 50 and 100 nMsiRNA concentrations. When the TGFβII receptor antibody was preincubatedwith the antigenic peptide before probing, the immunoreactive banddisappeared (FIG. 2, lane 7). The lack of a signal in this lanedemonstrates the specificity of the antibody. The turnover rate varieswith the presence of ligand binding and with the cell type used. Thehalf life of TGFβRII receptor varied from 2-6 hours. TGFβBII receptortranscript was examined by real time PCR and it was seen that the siRNAcompositions significantly changed the level of receptor mRNA.

Example 3 Reduction of Fibronectin Assembly and Secreted Fibronectin bysiRNAs

Using immunofluorescence, it was demonstrated that untreated controlcorneal fibroblasts exhibited robust fibronectin deposition and a densefibrillar network over cells. A similar pattern was also observed incells treated with scrambled siRNA. In these analyses immunofluorescenceof untreated fibroblasts or fibroblasts treated for 48 hours withscrambled siRNA, 100 or 200 nM NK1, or 100 or 200 nM SS1 was performedto visualize fibronectin matrix. Staining of nuclei was performed usingDAPI stain. These studies showed that fibronectin deposition wasmarkedly reduced in corneal fibroblasts 48 hours after transfection withboth 100 and 200 nM of NK1 and SS1 siRNAs. The nuclei werecounterstained by DAPI. The cell density was similar in the variousspecimens and thus the decreased fibronectin assembly was not related toa decrease in cell number.

The effects of the siRNAs on the fibronectin fibrillogenesis also wasexamined through observing changes in fibronectin secretion. Cornealfibroblasts, 48 hours after transfection, were incubated in serum-freemedium for 24 hours. Proteins collected in the media were subjected toWestern blotting. A 220-Kda fibronectin band was observed in allsamples. Consistent with the immunofluorescence data, treatment with 100and 200 nM NK1 and SS1 resulted in a decreased level of fibronectinsecreted into the culture media. The two siRNAs were equally effective,eliciting greater effect with 200 nM than 100 nM.

Example 4 Retardation of Cell Migration by siRNAs

Wound scratch assays indicated that corneal fibroblasts were able tomove into the wounded area. Within 7 hours, untreated control andscrambled RNA-transfected cells filled most of the pipette tip-generatedwound, covering 83.0±2.2% and 80.4±2.6% of the area, respectively. Bycontrast, the wound area covered by 100 and 200 nM NK1 and SS1transfected cells was significantly smaller (P<0.0001) varying from 37to 57%. The blockage of cell migration was more dramatic with the higherconcentration of siRNAs. Experiments were repeated 3 times yieldingsimilar results.

Example 5 Reduction of Inflammatory Response and Fibrosis in a MouseModel

A conjunctival scarring mouse model was generated by injecting phosphatebuffered saline (PBS) and latex beads into subconjunctival space.Inflammation response, as judged by the number of inflammatory cells intissue sections, was more severe on post-injection day 2 compared tothose obtained from eyes injected with PBS alone. The inflammatoryresponse observed on day 2 subsided on days 4 and 7.

NK1, SS1, and scrambled siRNAs were introduced into mouse eyes togetherwith phosphate buffered saline (PBS) and latex beads in a double maskedmanner. One eye of each mouse was treated with NK1 or SS1, and thecontralateral eye was treated with the scrambled RNA. Eyes in other micewere either left untreated or injected with PBS and latex beads alone toserve as controls. Two days following the injection, numerousinflammatory cells were observed underneath the conjunctival epitheliumin the scrambled RNA-treated and PBS/beads-injected control eyes. Theinflammatory cells were less in NK1 and SS1-treated eyes.

On post-injection days 7 and 14, the number of inflammatory cells wasreduced in all treated or injected eyes. The subconjunctival space inthe scrambled RNA-treated and PBS/beads-injected control eyes was filledwith fibroblasts. The density of conjunctival fibroblasts was higherthan that seen in eyes treated with NK1 or SS1. Picrocirius red stainingto demonstrate collagen deposition further showed that the fibroticresponse on day 14 was repressed by NK1 and SS1 siRNAs.

Example 6 Inhibition of TGFβRII Using siRNA on Endothelial Cells

Human umbical vein endothelial cells (HUVEC) were plated at 3×10⁻⁵cells/well and allowed to grow into confluent monolayers. The followingday, the cells were treated with TransIT-TKO reagent only (negativecontrol), scrambled siRNA oligonucleotides, NK1 siRNA oligonucleotidesand SS1 siRNA oligonucleotides, all in TransIT-TKO reagent. 200 nMconcentrations of the oligonucleotides was used, however, greater orlesser concentrations may be used. The cellular uptake of theoligonucleotides was demonstrated by fluorescence microscopy using theCy3-labeled luciferase. Images were taken 48 hours post RNAi treatment.

Immunofluorescence analyses showed that TGFβRII was distributeddiffusely in the cytoplasm of untreated control corneal fibroblasts(FIG. 4 a). In the presence of NK1 and SS1 siRNAs, the TGFβRII stainingintensity was dramatically reduced (FIG. 4 c and d; respectively). Theseresults are consistent with the experiments carried out in cornealfibroblasts described in Example 2. Cells treated with scrambled siRNA(FIG. 4, b) showed a similar intensity and pattern as the untreatedcontrol cells, demonstrating the specificity of NK1 and SS1 effects.

1-11. (canceled)
 12. An siRNA molecule that reduces expression of theTGFβ type II receptor, wherein the siRNA molecule is 19-25 base pairs inlength and targets at least a portion of the coding sequence of anucleic acid molecule comprising the nucleic acid sequence of SEQ IDNO:159.
 13. A composition comprising the siRNA molecule of claim 12 anda pharmaceutically acceptable carrier.
 14. The composition of claim 13,further comprising a wound healing agent.
 15. A method for promotingwound healing in a mammal comprising administering a therapeuticallyeffective amount of a composition comprising the siRNA molecule of claim12 to a mammal in need of treatment.
 16. A method for inhibitingfibrosis in a mammal comprising administering a therapeuticallyeffective amount of a composition comprising the siRNA molecule of claim12 to a mammal in need of treatment.
 17. A method for inhibitingangiogenesis in a mammal comprising administering a therapeuticallyeffective amount of a composition comprising the siRNA molecule of claim12 to a mammal in need of treatment.
 18. A method for preventingglaucoma in a mammal comprising administering to a mammal in need oftreatment a therapeutically effective amount of a composition comprisingthe siRNA molecule of claim
 12. 19. A method of preventing restenosis ina mammal comprising administering to said mammal a therapeuticallyeffective amount of a composition comprising the siRNA molecule of claim12.
 20. A method of preventing or treating scarring in a mammalcomprising administering to said mammal a therapeutically effectiveamount of a composition comprising the siRNA molecule of claim 12.